Impact of ompk36 genotype and KPC subtype on the in vitro activity of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam against KPC-producing K. pneumoniae clinical isolates

Abstract Objectives The availability of new β-lactam/β-lactamase inhibitors ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam have redefined contemporary treatment of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) infections. We aimed to characterize and contrast the in vitro activity of these agents against genetically diverse KPC-Kp clinical isolates. Methods We analysed genomes of 104 non-consecutive KPC-Kp isolates and compared the in vitro antibiotic activity by KPC subtype and ompK36 genotype. MICs were determined in triplicate by CLSI methods. Twenty representative isolates were selected for time–kill analyses against physiological steady-state and trough concentrations, as well as 4× MIC for each agent. Results Fifty-eight percent and 42% of isolates harboured KPC-2 and KPC-3, respectively. OmpK36 mutations were more common among KPC-2- compared with KPC-3-producing Kp (P < 0.0001); mutations were classified as IS5 insertion, glycine-aspartic acid insertion at position 134 (GD duplication) and other mutations. Compared to isolates with WT ompK36, ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam MICs were elevated for isolates with IS5 by 2-, 4- and 16-fold, respectively (P < 0.05 for each). Against isolates with GD duplication, imipenem/relebactam and meropenem/vaborbactam MICs were increased, but ceftazidime/avibactam MICs were not. In time–kill studies, ceftazidime/avibactam-mediated killing correlated with ceftazidime/avibactam MICs, and did not vary across ompK36 genotypes. Imipenem/relebactam was not bactericidal against any isolate at trough concentrations. At steady-state imipenem/relebactam concentrations, regrowth occurred more commonly for isolates with IS5 mutations. Log-kills were lower in the presence of meropenem/vaborbactam for isolates with GD duplication compared with IS5 mutations. Conclusions Our investigation identified key genotypes that attenuate, to varying degrees, the in vitro activity for each of the new β-lactam/β-lactamase inhibitors. Additional studies are needed to translate the importance of these observations into clinical practice.


Background
Infections due to carbapenem-resistant Enterobacterales (CRE) are a major cause of attributable death worldwide. 1 In the USA alone, CRE infections are responsible for over 13 ,000 hospitalizations, 1,100 deaths, and $130 million in healthcare costs each year. 2 The most common CRE pathogen in US-based surveillance studies is Klebsiella pneumoniae, the majority of which harbour K. pneumoniae carbapenemase (KPC) enzymes. 3,4 Accordingly, KPC has been a primary target for drug discovery efforts, leading to the development of three novel β-lactamase inhibitors that inhibit KPC: avibactam, relebactam and vaborbactam. Avibactam and relebactam are diazabicyclooctane β-lactamase inhibitors that have been partnered with ceftazidime and imipenem, respectively. Vaborbactam is a cyclic boronic acid β-lactamase inhibitor paired with meropenem. The resulting combinations of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam have each shown potent in vitro activity against KPC-producing K. pneumoniae (KPC-Kp), [5][6][7] and favourable clinical outcomes for patients infected with carbapenemresistant pathogens. [8][9][10][11] Each are now recommended as preferred treatment options for the management of patients with CRE infections due to KPC-producing Enterobacterales. 12,13 Which agent should be prioritized for KPC-Kp specifically, however, is still unclear.
Carbapenem resistance in KPC-Kp is predominantly mediated by KPC; however, the loss or modification of major porin channels in the outer cell membrane can contribute to high-level resistance. 14,15 The two major porins in K. pneumoniae are OmpK35 and OmpK36, which are encoded by ompK35 and ompK36, respectively. Loss-of-function mutations in ompK35 are common among KPC-Kp. 7,16 ompK36 genotypes, on the other hand, vary significantly among clinical isolates. Two major mutations have been described in KPC-Kp. The first in an IS5 insertion element within the ompK36 gene promoter or coding sequence that is associated with decreased ompK36 expression. 14,17 The second is a 6 bp insertion in the L3 loop encoding a glycine and aspartic acid. This insertion is known to constrict the inner pore diameter and subsequently limit intake of nutrients as well as antibiotics like the carbapenems. 15,16 It is unclear if other reported mutations or insertions in ompK36 impact the activity of carbapenems, or more specifically the activity of ceftazidime/avibactam, imipenem/relebactam or meropenem/ vaborbactam.
The available data show that various molecular mechanisms can contribute to the in vitro activity of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam. Translating these data to inform clinical decisions is a particular challenge because all three agents are likely to be reported as susceptible for the vast majority of KPC-Kp. We hypothesized that the underlying molecular mechanisms of resistance in KPC-Kp contribute to the in vitro activity of these agents to varying degrees. The objective of this study was to compare the in vitro activity and killing effects for ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam against genetically diverse KPC-Kp clinical isolates.

Characterization of Kpc-Kp clinical isolates
Clinical isolates collected from patients not previously treated with ceftazidime/avibactam, imipenem/relebactam or meropenem/vaborbactam were selected from local biorepositories. All isolates were stored at −80°C and subcultured twice on Mueller-Hinton agar (MHA; Becton, Dickinson, & Company, Sparks, MD, USA) prior to testing. WGS was performed on an Illumina platform as described previously. 18 Species, ST and KPC subtypes were confirmed with WGS analyses. Core-genome SNPs were identified using pairwise comparisons with Snippy (https:// github.com/tseemann/snippy). Genotypes for ompK35 were categorized as either WT or truncated. Genotypes for ompK36 were denoted as WT or mutant. Mutant ompK36 genotypes included IS5 (IS5 insertion element in either promoter region or ompK36 coding sequence), GD duplication (glycine and aspartic acid insertions at amino acid positions 134 and 135) or other (one or more divergent sequences, insertions, substitutions or deletions) mutations. WT ompK35 and ompK36 genes were defined as described previously. 14,15,19

Susceptibility testing
MICs were determined in triplicate by standardized broth microdilution methods; susceptibility was defined according to CLSI interpretive criteria. 20 Tested concentrations of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam ranged from 0.12-256, 0.06-64 and 0.008-8 mg/L, respectively. β-Lactamase inhibitors avibactam, relebactam and vaborbactam were tested as fixed concentrations of 4, 4 and 8 mg/L, respectively. Quality control was assessed using Pseudomonas aeruginosa ATCC 27853 and K. pneumoniae ATCC 700603, and results were reported only when MICs for control strains were within acceptable ranges.

In vitro killing activity
Time-kill assays were performed on 20 isolates that were representative of the predominant KPC subtypes and ompK36 genotypes. Each isolate was grown overnight in CAMHB (Becton, Dickinson, & Company, Sparks, MD) at 37°C with shaking. Experiments were performed in 8 mL of CAMHB using an initial inoculum of 1 × 10 6 cfu/mL. Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam were tested at a concentration of 4× MIC. In addition, physiological free drug steady-state and trough concentrations were identified from published studies. [21][22][23] Steady-state exposures included 32, 4 and 8 mg/L for ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam, respectively. Corresponding trough concentrations were 4, 0.5 and 1 mg/L, respectively. Simulated trough concentrations were selected to represent the lowest exposure achieved in patients prior to administration of the next dose. All experiments were incubated at 37°C with 200 rpm shaking. Samples were taken at 0, 2, 6, 10 and 24 h, serially diluted, plated on Mueller-Hinton agar plates, and incubated overnight at 37°C. Colonies were enumerated and reported as cfu/mL; bactericidal activity was defined as ≥3 log kill after 24 h of incubation compared with the starting inoculum.

Statistical analysis
Categorical and continuous variables were analysed using a chi-squared (or Fisher's exact) test and Mann-Whitney U-test, respectively. Mean logkills after 24 h were compared using a Student's t-test. A two-tailed P value of ≤ 0.05 was considered statistically significant.
Next, 20 isolates were selected for time-kill analyses; the median core-genome SNP difference across isolates was 76 (range: 2-18, 291). Isolates were selected to represent each of the four predominant KPC-Kp subgroups associated with MIC differences. Five isolates from each subgroup of KPC-2 with WT ompK36, KPC-3 with WT ompK36, KPC-2 with IS5, and KPC-2 with GD duplication were included. Corresponding within-group median coregenome SNP differences were 46, 128, 50 and 33, respectively (Table S2).
Imipenem/relebactam was bactericidal against 90% of isolates at steady-state concentrations (4 mg/L), demonstrating KPC-2 (n=60) WT Table S1. mean log-kills of −4.77 cfu/mL across all isolates. In contrast, trough concentrations (0.5 mg/L) were not bactericidal against any isolate ( Figure 5). In the presence of imipenem/relebactam concentrations of 4× MIC, mean log-kills against isolates with WT, IS5 and GD duplication ompK36 genotypes were −1.48, −2.69 and −3.57 cfu/mL, respectively. There was no difference in mean log-kills or rates of bactericidal activity for isolates with IS5 or GD duplication genotypes; however, bacterial regrowth was noted for isolates with IS5 mutations, but not GD duplication against steady-state concentrations ( Figure 6)  isolates with IS5 mutations (−5.71 cfu/mL) compared with GD duplication (0.32 cfu/mL; P = 0.002); regrowth after 10 h was observed against GD duplication, but not IS5 genotypes.

Discussion
In this study, we compared the MICs and in vitro killing activity of ceftazidime/avibactam, imipenem/relebactam and meropenem/ vaborbactam against KPC-Kp clinical isolates with the most common KPC subtypes and ompK36 genotypes encountered clinically. Our data demonstrate several key differences between the agents which may help differentiate front-line treatment options for KPC-Kp infections. First, we show that MICs for each agent were elevated against isolates harbouring IS5 mutations in ompK36. Such mutations increased the median MICs (compared with WT) by 2-, 4-and 16-fold for ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam, respectively. While the magnitude of MIC change was greatest for meropenem/vaborbactam, rates of in vitro susceptibility did not change (Table 1). In contrast, 28% of KPC-Kp with IS5 mutations in ompK36 were categorized as non-susceptible to imipenem/relebactam. These findings align with attenuated killing and regrowth during time-kill studies in the presence of physiological trough and steady-state imipenem/relebactam concentrations against KPC-Kp with IS5 mutations (Figure 6). We did not identify differences for the in vitro killing activity of ceftazidime/avibactam against isolates with or without IS5 mutations, suggesting that this agent was the least impacted of the three agents tested. Median imipenem/relebactam and meropenem/vaborbactam, but not ceftazidime/avibactam, MICs were also increased against KPC-Kp harbouring a GD duplication in ompK36 (Figure 3). At simulated steady-state concentrations, imipenem/relebactam and meropenem/vaborbactam were bactericidal against 100% and 80% of isolates harbouring GD duplications, respectively; however, at trough concentrations, corresponding rates of bactericidal killing were reduced to 0% and 20%, respectively. In fact, trough concentrations of imipenem/relebactam were not bactericidal against any KPC-Kp isolate studied. These data suggest that each major ompK36 mutation has a differential impact on ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam. Other less common mutations in ompK36 did not impact MICs overall; however, two isolates demonstrating elevated meropenem/vaborbactam and imipenem/relebactam MICs were noted. One isolate harboured a glycine to alanine substitution 11 bp upstream of ompK36, and the other showed an amino acid insertion at position 104 resulting in a frameshift (Table S1). Finally, we noted paradoxical killing effects with imipenem/relebactam and meropenem/vaborbactam concentrations at 4× MIC, such that log-kills were greater against isolates with elevated MICs (Figure 5). We anticipate that greater killing of isolates harbouring ompK36 mutations was due to a potential fitness cost, or alternatively selection of a resistant subpopulation for isolates with WT genotypes at baseline. Our findings corroborate and extend previous reports demonstrating the impact of KPC subtypes and ompK36 mutations on the activity of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam. [5][6][7]24 We have identified key genotypes that attenuate, to varying degrees, the activity for each agent. For ceftazidime/avibactam, MICs are 2-fold higher for KPC-3 compared with KPC-2-producing isolates. At the same time, ceftazidime/avibactam was the least impacted of the three agents by mutations in ompK36 where only IS5 mutations resulted in a 2-fold MIC increase, but no change in the killing activity for any ceftazidime/avibactam concentration tested in time-kill studies.
Next, we found that imipenem/relebactam MICs increased in a stepwise manner across ompK36 genotypes (Figure 3). Median MICs increased from 0.25 to 0.5 to 1 mg/L against KPC-Kp with ompK36 WT, GD duplication and IS5 insertion, respectively. In time-kill studies, we noted less killing against isolates with IS5 mutations when compared with isolates with GD duplication or WT ompK36 genotypes ( Figure 6). IS5 insertions in ompK36 decrease ompK36 expression and have been previously validated as a cause of elevated imipenem/relebactam MICs. 16,25 Imipenem/relebactam MICs were less impacted by isolates that harboured a GD duplication in ompK36, and in vitro killing was similar among isolates with either WT or GD duplication genotypes. We propose the activity of imipenem/relebactam is  Impact of ompk36 on KPC-Kp susceptibility to novel agents By comparison, the in vitro activity of meropenem/vaborbactam appears to be significantly impacted by GD duplications, resulting in a 16-fold median MIC increase and less killing during time-kill experiments. Increased MICs were also identified against isolates with IS5 genotypes. Against isolates with either GD duplication or IS5 mutations, however, median meropenem/vaborbactam MICs were increased to 0.5 mg/L, which remains well below the CLSI susceptibility breakpoint. Based on pharmacokinetic/pharmacodynamic (PK/PD) modelling, meropenem/vaborbactam would be predicted to be effective even at the highest MICs identified in our study. 28 This aligns with our time-kill data that showed meropenem/vaborbactam concentrations of 8 mg/L were bactericidal across ompK36 genotypes. Moreover, the frequency of mutant selection at physiological exposures appears to be low. 29 To date, only two cases of meropenem/vaborbactam treatment-emergent non-susceptibility have been described, and ompK36 mutations were implicated in both. 19,26 Like meropenem, vaborbactam accesses the periplasmic space through OmpK36, 30 and thus both agents are reliant upon intact porin channels for cell entry.
Comparative clinical data for ceftazidime/avibactam and meropenem/vaborbactam are limited, 31 and have not been published for imipenem/relebactam. Among patients with KPC-Kp infections, rates of treatment-emergent resistance are highest for ceftazidime/avibactam. 19,[31][32][33] Resistance to ceftazidime/avibactam is mediated by mutations in the bla KPC Ω-loop that result in preserved in vitro activity against meropenem/vaborbactam and imipenem/relebactam (Table S3). 5,6,34 Isolates resistant to meropenem/vaborbactam generally demonstrate crossresistance to imipenem/relebactam, but not ceftazidime/avibactam. 19,26 Interestingly, during in vitro selection studies with meropenem/vaborbactam, isolates with WT ompK36 were more likely to select ompK36 mutations than isolates with mutant ompK36 at baseline. In the latter group, isolates were found to have an increased bla KPC copy number following in vitro exposure to meropenem/vaborbactam. 29 Similar observations have been noted for imipenem/relebactam resistance, where bla KPC copy number and loss-of-function ompK36 mutations are selected in vitro. 16 A key difference between the agents is that mutations in bla KPC refractory to relebactam inhibition were selected with imipenem/relebactam, but not meropenem/vaborbactam. 16,29 The clinical impact of mutations in ompK36 on the efficacy of ceftazidime/avibactam, imipenem/relebactam and meropenem/ vaborbactam remains unclear. Animal models of ompK36 mutant strains have demonstrated attenuated virulence, 15,35 and porin genotypes are typically not characterized in clinical outcome-based studies. 19,32 For now, clinicians must make decisions based on the available clinical and preclinical data. Ceftazidime/avibactam is the least impacted by ompK36 mutations; however, selection for ceftazidime/avibactam resistance through bla KPC mutations is a potential threat to the agent's utility for KPC-Kp infections. The presence of ompK36 mutations, particularly IS5 insertions, elevates imipenem/relebactam MICs resulting in a proportion of isolates that are categorized as nonsusceptible. In our study, simulated physiological imipenem/relebactam trough and steady-state exposures did not durably inhibit growth of KPC-Kp across ompK36 genotypes. Importantly, clinical data are very limited for imipenem/relebactam when compared with ceftazidime/avibactam or meropenem/vaborbactam. 11,36 Finally, we found that meropenem/vaborbactam MICs were elevated by GD duplication and IS5 mutations in ompK36; however, median MICs were well below the current susceptibility breakpoint and within the range of optimal PK/PD target attainment. 28,29 Taken together, our in vitro findings provide support for meropenem/vaborbactam as the preferred agent for treatment of KPC-Kp infections.
Some limitations with the current analysis should be acknowledged. First, all isolates were collected from patients at a single centre, and despite selecting strains that were genetically diverse, not all KPC-Kp genotypes were represented. Most notably, we did not study KPC-3-producing isolates that harboured GD duplications; however, comparable findings for KPC-3 have been reported previously. 26,27,29 We also did not fully characterize ompK36 mutations that were classified as 'other' mutations. Although ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam MICs were generally not impacted unless GD duplication or IS5 mutations were present, it does not preclude the possibility that other loss-of-function mutations in ompK36 may manifest in a similar impact on these agents. In addition, we did not investigate the in vitro activity against KPC variants selected by ceftazidime/avibactam treatment as part of this study, but have in prior publications. 5,6,24 For completeness, we have summarized these data in Table S3. Secondly, our time-kill studies were limited to 24 h experiments to investigate initial killing effects, but not the presence of resistant subpopulations. In doing so, we have cautiously interpreted our findings with acknowledgement that dynamic drug exposures over longer treatment courses could yield different results. Indeed, humanized ceftazidime/avibactam exposures in a hollow-fibre infection model against KPC-Kp with varying genetic backgrounds may respond differently. 37 Despite these limitations, our results shed new light on the specific impact of ompK36 mutations on each of the recently approved β-lactam/ β-lactamase inhibitor combinations. Future studies are needed to investigate comparative rates for the frequency of mutant selection, and the impact of the genetic background on treatment efficacy both in vitro and in vivo.